Preclinical Assessment in Transgenic NOD Mice of a Novel Immunotherapy for Type 1 Diabetes: Lipoplexes Down-Modulate...

The C1858T PTPN22 (R620W) variant has been implicated in the pathogenesis of several autoimmune disorders and represents a promising immunotherapeutic target for Type 1 diabetes. We have been implementing a novel immunotherapeutic approach based on the use of lipoplexes that deliver siRNA duplexes. The efficacy and safety of lipoplexes was previously demonstrated in vitro in halting variant expression in the peripheral blood of patients. Preclinical safety and efficacy must be ascertained in vivo in appropriate animal models before clinical investigations can be undertaken, according to regulatory authorities in Europe. In the light of the foregoing, this study aims to verify that lipoplexes against the murine Ptpn22-R619W, equivalent to the human PTPN22-R620W, could be used for animal experimentation. The murine fibroblast cell line L929 was transfected with the PF62-pLentiPtpn22-R619W plasmid. We designed specific siRNA duplexes for the Ptpn22-R619W allele and formulated them into cationic lipoplexes in order to halt variant expression in the transfected L929 cell line. Transfection of fibroblasts expressing R619W using lipoplexes resulted in efficient silencing at 100 pmol siRNA after 48 h post-transfection, reaching higher significant knockdown after 72 h. Lipoplexes efficiently suppress pathogenic Ptpn22 variant expression in vitro, supporting the feasibility of a pre-clinical platform for testing of in vivo lipoplexes in CRISPR-engineered NOD/ShiLtJ mice carrying the R619W mutation.